Salmonella enterica sv. Typhi (S. Typhi) is the etiologic agent of typhoid fever in humans and can cause life-threatening infection in normal hosts. Current typhoid vaccines are only moderately immunogenic and protective. Improvements in vaccine technology have been constrained by the lack of a small animal model. S. Typhi is highly adapted to humans and fails to cause progressive infection in normal mice. S. Typhimurium- murine interactions are used to model S. Typhi-human interactions, but murine typhoid and human typhoid exhibit major differences in both pathogen and host determinants. In the application, we report a breakthrough in the development of a small animal model for the study of human typhoid fever, using NOD-scid IL2rgamma null mice engrafted with CD34+ human hematopoietic stem cells from T-cell depleted umbilical cord blood. In preliminary studies, we have shown that S. Typhi Ty2 can replicate in vivo within engrafted mice following intraperitoneal inoculation to cause lethal infection that recapitulates crucial features of human typhoid. In contrast, S. Typhi is unable to cause productive infection in non-engrafted NOD-scid IL2rgamma null animals or immunocompetent NOD controls. This application describes a pilot project to evaluate this new model and rigorously test the hypothesis that S. Typhi requires human mononuclear cells to replicate in vivo. The specific aims of the project are: 1.Chacterization of the Humanized Mouse Typhoid Model. The novel model will be characterized using microbiologic, histopathologic, and flow cytometric methods to define the course of infection, innate immune response, and importance of apoptosis. 2. Identification of S. Typhi Genes Required for Replication in Humanized Mice. A microarray-based method will be used to identify transposon mutations that impair S. Typhi virulence. Our model provides an unprecedented opportunity to gain new insights into typhoid pathogenesis, identify new S. Typhi vaccine candidates and devise new strategies for the prevention of S. Typhi infections. PUBLIC HEALTH RELEVANCE: The goals of this proposal are to characterize a novel humanized mouse model of human typhoid and to use this model to identify genetic determinants of Salmonella Typhi virulence. A small animal model that mimics human typhoid represents a major advance toward the development of more effective typhoid vaccines. Public Health Statement: Salmonella Typhi remains a serious human pathogen, estimated to cause 16,000,000 infections each year and resulting in more than 600,000 deaths. The two currently available typhoid vaccines are not completely effective and provide only short-term protection. Typhoid vaccine development has been constrained by the lack of a small animal model. In this application, we report the breakthrough development of a humanized mouse model that recapitulates critical aspects of human typhoid. Salmonella Typhi is able to replicate in mice engrafted with human immune cells obtained from umbilical cord blood, producing a progressive lethal infection. For the first time, this provides an experimental system to analyze the host and microbial determinants of clinical outcomes in typhoid fever. The studies described in this proposal will provide new insights into the interaction of S. Typhi with the human immune system and identify new strategies for the construction of safe and effective typhoid vaccines.